Although much of the improvement in outcomes from acute lung injury has been ascribed to lung-protective mechanical ventilation strategies, ventilator-induced lung injury (VILI) remains an important element of morbidity and mortality in the critically ill patient. Using an unbiased genome-wide association study, we (Li et al, Am J Resp Cell Mol Biol 2012) identified a WNT1-inducible signaling pathway protein 1 (WISP1) in murine VILI. Moreover, we and others have identified that innate immune signaling via TLR4 plays a critical role in the pathogenesis of VILI and that stretch-induced WISP1 expression and its pro-inflammatory effect were TLR4- dependent. Accordingly, we propose: Specific Aim 1. To determine the molecular pathway by which mechanical stretch is coupled to respiratory epithelial WISP1 biosynthesis and VILI. We will: a) use pharmacological and genetic approaches to dissect contribution of non-canonical Wnt signaling pathway in WISP1 biosynthesis in cyclic stretched cultured murine respiratory epithelium; b) use biochemical determinants to associate non-canonical pathway in intact lung of mice after HTV (12 ml/kg x 6h); and c) determine the contribution of epithelial derived WISP1 in VILI by changes in alveolar capillary permeability after HTV in wildtype, WISP1 -/- and after silencing WISP1 in alveolar macrophages in situ with intratracheal lentiviral delivery of shRNA (and hence leaving epithelial derived WISP1 as sole source). Specific Aim 2. To determine the role of WISP1 in communicating mechanical stress responses to innate immune system leading to VILI: We will determine the contribution of macrophage (and neutrophil) TLR4 signaling in VILI by comparing the effect of HTV on wildtype, whole body TLR4 and myeloid-cell-specific TLR4 null mice (Lyz-TLR4; Nace et al, Hepatology 2013). Specific Aim 3. To determine the molecular determinants by which WISP1 acts as an accessory molecule transducing stress of mechanical stretch in airway epithelium to pro-inflammatory phenotype of macrophages via TLR4. We will define requisite components for proinflammatory (e.g. TNF; NF?B) effect of WISP1 using peritoneal macrophages and confirm accrued information in primary murine alveolar macrophage cultures (that are limiting in number) from wildtype and CD14 and 3 and 5 null mice. We will then determine the obligatory roles of 3 and 5 in WISP1 mediated VILI by contrasting the effect of HTV on 3 and 5 null mice, with and without i.t. injection of WISP1. Collectively, these studies will provide novel insight into the role of matricellular protein, WISP1, and the Wnt pathway, in transducing the effect of mechanical stress on respiratory epithelium to innate immune system and role of TLR4 activation of alveolar macrophages in pathogenesis of VILI. Such mechanistic insight may lead to biomarkers, therapeutic targets in prevention or mitigation of VILI and further understanding of genetic determinants of susceptibility to VILI.